1. A cDNA for a CAKbeta/PYK2-binding protein (CBP-1) was cloned by screening a human brain cDNA library with the CAKbeta C-domain as a probe. CBP-1 was the human homologue of Hic-5. Our CBP-1 cDNA provided an evidence that Hic-5 has more than 16 additional amino acid residues at its N-terminal region in addition to the sequence previously described for mouse Hic-5. Hic-5 localizedatfocal adhesions. Hic-5 has 4 LIM domains and 5 LD motifs and is most closely related to paxillin. Hic-5 bound to the C-terminal half of the CAKbeta C-domain with its N-domain. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. When WFB cells were stimulated to enhance the tyrosine-phosphorylation of CAKbeta, the tyrosine-phosphorylation of Hic-5 was also enhanced.2. In COS-7 cells, the tyrosine-phosphorylation of Hic-5 was enhanced by co-expression of CAKbeta and was further enhanced by stimulating the coexpressed cells with osmotic-stress. The tyrosine-60 residue of Hic-5 was the major site of the tyrosine-phosphorylation because the Y6OF mutant of Hic-5 was nottyrosine-phosphorylated. The CAKbeta mutant deleted at the Hic-5 binding site was defective in the tyrosine-phosphorylation of Hic-5. The tyrosine-phosphorylated Hic-5 was bound to the SH2 domain of Csk but not to the SH2 domains of Fyn, Src, and Crk.3. The wild type CAKbeta localized in cytoplasm. A single amino acidsubstituted mutant of CAKbeta designated "A mutant" was found to localize exclusively in nucleus. A translocation of wild type CAKbeta to nucleus was observed under certain culture conditions. A portion of Hic-5 was also translocated to nucleus when coexpressed with the A mutant of CAKbeta.